a. Search for XMRV in prostate tumors. Several recent papers reported the detection of a new MLV, xenotropic murine leukemia virus-related virus (XMRV), in human prostate tumors (e.g., Urisman et al., PLoS Pathog. 2:e25, 2006; Schlaberg et al., PNAS 106:16351-16356, 2009). In collaboration with Drs. Karen Sfanos and Angelo De Marzo (Johns Hopkins School of Medicine), we surveyed nearly 800 prostate tumors for XMRV, using a combination of duplex real-time PCR and immunohistochemistry (IHC). Our studies used 22Rv1 cells, which are known to be infected with XMRV (Knouf et al., J. Virol. 83: 7353-7356, 2009), as positive controls, and uninfected human cells, such as 293T or HeLa cells, as negatives. Every PCR well was simultaneously analyzed for viral sequences and for CCR5, a single-copy human gene, as a test of the quality and quantity of sample DNA. Dilution of 22Rv1 DNA into HeLa DNA showed that we could detect the XMRV sequences in 1.5 22Rv1 cells, even in the presence of a 10,000-fold excess of HeLa cells. Our IHC assays used a rabbit antiserum raised against HPLC-purified Moloney MLV p30CA and another against HPLC-purified Moloney MLV gp70SU. Controls included not only 22Rv1, but also 293T cells transfected with a molecular clone of XMRV or mock-transfected. In all cases, the 22Rv1 cells and the XMRV-transfected cells stained with both antisera whereas the negative cells did not. Neither of these well-controlled assays gave any indication of XMRV infection in any of the prostate tumor samples. We concluded (Aloia et al., Cancer Res. 70:10028-10033, 2010) that the positive reports from other laboratories likely represented false positives. For example, positive PCR results can result from contamination with minuscule amounts of mouse DNA, and positive IHC results can arise if the antisera react with host-cell, as well as viral, antigens. Subsequent developments have revealed that XMRV was originally formed during xenotransplantation of the 22Rv1 tumor line, and that humans are almost certainly not infected by this virus (Paprotka et al., Science 333:97-101, 2011; Sfanos et al., Nat. Rev. Urol. 9:111-118, 2012). b. Development of a new MLV infectivity assay and further applications of the iGLuc infectivity assay strategy. As part of our continuing studies on XMRV, we adapted a retrotransposition assay (Curcio and Garfinkel, PNAS 88:936-940, 1991; Heidmann et al., PNAS 85:2219-2223, 1988) for detection of replication-competent gammaretroviruses. In this assay (called iGLuc), an MLV-based vector is placed into 293 cells. The vector carries a Gaussia luciferase gene in reverse orientation, interrupted by an intron in forward orientation. Only when this vector has been rescued by a competent helper virus and has progressed through the entire retroviral life cycle is the Gaussia luciferase expressed. This work was performed in collaboration with Drs. Gisela Heidecker, Vineet KewalRamani, and the late David Derse (HIV Drug Resistance Program, National Cancer Institute; see Mazurov et al., PLoS Pathog. 6:e1000788, 2010). Although other reporters can be used in this assay, the Gaussia luciferase offers unique advantages: it is extremely bright, rendering the assay very sensitive, and it is secreted, so that the assay can be performed directly on culture fluids. It is easily performed in a 96-well format. Together with Drs. Sfanos and De Marzo, we screened 70 widely used cell lines for the presence of MLVs, using both PCR and IHC assays (Sfanos et al., PLoS One 6:e20874, 2011). Three lines were found to be producing replication-competent MLVs. The viruses were characterized in considerable detail: two were found to be derived from the endogenous MLV Bxv-1, whereas the third was related to another xenotropic MLV. Their replication-competence was confirmed by the iGLuc assay. In some cases, the MLVs infected the human cells during the xenotransplantation passages used to establish the cell lines; in others, the infectionsprobably represent cross-contamination that occurred in other laboratories. We believe that the iGLuc strategy for detection of retroviral replication can have wide application. We have collaborated with Dr. Kenneth Cornetta (Indiana University) to demonstrate the utility of the iGLuc assay for testing gene-therapy vector preparations for replication-competent gammaretroviruses (Aloia et al., Gene Ther. 20:169-176, 2013), and we intend to do the same for HIV-1-based gene-therapy vectors. c. Characterization of an HIV-1 entry inhibitor. Several years ago, we and colleagues at the NCI-Frederick described a small-molecule entry inhibitor of HIV-1 entry, named Stibavirin or NSC13778 (Yang et al., J. Virol. 79:6122-6133, 2005). This compound is already FDA-approved for other uses. To gain further insight into its mechanism of action, Dr. John Mellors (University of Pittsburgh) has now selected HIV-1 mutants partially resistant to the drug. We have shown that the changes he observed in and near the V3 loop of gp120 are responsible for this resistance. Several kinds of data show that Stibavirin blocks entry by binding to CD4, preventing gp120 from binding. It seems possible that this type of inhibition could be a useful addition to antiviral therapy. The mechanism of the resistance is being analyzed by molecular modeling in collaboration with Drs. Rick Gussio and Tam Nguyen (Developmental Therapeutics Program, National Cancer Institute). d. Inactivation of retroviruses by lectins. In collaboration with Dr. Kathy Jones (CCR), we surveyed a series of gamma- and delta-retroviruses for their sensitivity to inactivation by several lectins. The pattern of responses was found to be complex. e. Testing breast cancer samples for retroviruses. In collaboration with Dr. Edward Gabrielson (Johns Hopkins Medical Institution), we have developed sensitive assays for the presence of mouse mammary tumor virus-related sequences in human breast cancers. Patents Linked to Project: U.S. Patent #7,572,828: Identification of Anti-HIV Compounds Inhibiting Virus Assembly and Binding of Nucleocapsid Protein to Nucleic Acid; issued August 11, 2009; Robert Shoemaker, Michael Currens, Alan Rein, Ya Xiong Feng, Robert Fisher, Andrew Stephen, Shizuko Sei, Bruce Crise, Louis Henderson, and Karen Worthy. This patent describes a class of compounds with anti-HIV-1 activity, which are under investigation for use in antiretroviral therapy. Patent pending: 03773233.6 (EP application). [Corresponds to Rein Project 4 in the October 2011 site visit report of the HIV Drug Resistance Program]